Effects Of Microcystin-LR On Downstream Targets Of Protein Phosphatase 2A In A Human Liver Cell Line-HL7702

The outbreak of water blooms caused by increasingly severe eutrophication not only damages aquatic organisms but also affects human health through the consumption of contaminated aquatic products, agricultural plants or drinking water. Microcystins are a family of monocyclic heptapeptides produced by cyanobacteria during water blooms. Microcystins are too stable to degrade naturally and it is difficult to remove them from water. Microcystins are of increasing importance due to their toxicity and potent tumor promoting activity, so the exact mechanisms of microcystin toxicity need further investigation.The basic structure of microcystins is composed of five fixed amino acids and two variable amino acids. There are more than 80 variants of microcystins being identified including amino acid variations and modifications. Microcystin-LR (MC-LR) is the most common as well as the most toxic one among these, with two variable amino acids, Leu and Arg. The liver is the organ most affected by exposure to MC-LR. Acute exposure of MC-LR leads to intrahepatic hemorrhage or hepatic insufficiency. Chronic exposure to lower concentrations of MC-LR is likely related with some illnesses or cancers. A group of hepatocyte uptake transporters termed organic anion transporting polypeptides (OATPs) present mainly in liver are crucial for MC-LR transferring into cells. Microcystins also concentrate in organs such as kidney, colon, reproductive system and nervous system, therefore the illnesses attributed to microcystin intoxication are gastroenteritis and related diseases, allergic and irritation reactions.Studies on toxic mechanisms of MC-LR have revealed that MC-LR exposure induces the alteration of the cytoskeleton, cell apoptosis, and the production of reactive oxygen species (ROS) and activates cellular signal transduction. MC-LR has been recognized as the strong inhibitor of PP2A, which may be the major reason of the implicated intoxication mechanism of MC-LR. However, after MC-LR enters the cell, the effect of microcystin-LR on downstream targets of protein phosphatase 2A, either adaptive or cytotoxic, are not quite clear. Although a number of MC-LR target proteins have been identified using proteomic and phosphoproteomic approaches, these studies can not dynamically reflect the relationship between PP2A and the cellular effects. And few studies of the effects of microcystins on human hepatocytes have been conducted.To further reveal the possible cellular effects and the relationship between PP2A and the cellular effects induced by MC-LR, we use a human liver cell line-HL7702 to investigate the effects of microcystin-LR on downstream targets of PP2A. We also try to establish a network consisted of PP2A and its targets, which may provide more evidences for the mechanisms of MC-LR toxication.The present study is consists of two parts. The first part is to examine the cellular effects induced by MC-LR, which includes:â‘ the alteration of PP2A activity by MC-LR;â‘¡the alteration of MAPKs'phosphorylation by MC-LR;â‘¢the alteration of cytoskeleton by MC-LR;â‘£the alteration of cell cycle and cell viability by MC-LR. The second part is to confirm the relationship among different cellular effects, which contains:â‘ the influence of D-erythro-Sphingosine(PP2A activator) on MC-LR-induced protein phosphorylation;â‘¡the influence of MAPK inhibitors on downstream targets.The results were shown as belowThe first part:1. MC-LR binded with PP2A/C subunit and inhibited PP2A activity in HL7702 cell.2. MC-LR markedly upregulated the phosphorylation of MAPKs in a time- and concentration-dependent manner. The ERK and JNK were more quickly phosphorylated than p38 MAPK. 3. MC-LR induced the alteration of cytoskeleton:â‘ MC-LR induced the reassembly of microfilaments and microtubules;â‘¡MC-LR decreased the acetylated-tubulin expression;â‘¢MC-LR altered distribution of tyrosinated-tubulin in cell.4. MC-LR induced the hyperphosphorylation of Tau, HSP27, VASP; MC-LR influenced the distribution of Tau, HSP27.5. MC-LR altered the phosphorylation of p53, cdc2, cdc25c, but had no effect on cell cycle.6. MC-LR exposure had no effect on cell viability in the present study. The second part:1. DES(PP2A activator)partially blocked MC-LR-induced phosphorylation of Tau, HSP27. DES partially blocked phosphorylation of p53, cdc25c, cdc2.2. MC-LR induced colocalization of B56 a and p-cdc25c, and the colocalization was in the cortical part beneath the cell membrane.3. The phosphorylation of HSP27 was suppressed by inhibitor of p38 MAPK and JNK. The phosphorylation of Tau was suppressed by p38 MAPK inhibitor.The conclusions are shown as below:1. MC-LR binds with PP2A/C subunit and inhibits PP2A activity in HL7702 cells exposed to MC-LR.2. MC-LR could activate the classic MAPK pathways-p38 MAPK, ERK and JNK pathway. And the sensitivity of p38 MAPK to MC-LR is lower than ERK and JNK.3. MC-LR decreases cytoskeleton stability by decreasing acetylated-tubulin, altering distribution of tyrosinated-tubulin in the cell, inducing the reassembly of microfilaments and microtubules, inducing the hyperphosphorylation of Tau by p38 MAPK as a result of PP2A inhibition, and the phosphorylation of Tau may destabilize the cytoskeleton. Besides, MC-LR induces HSP27 phosphorylation by p38 MAPK and JNK as a result of PP2A inhibition and the phosphorylation of HSP27 may be against the further damage.4. MC-LR influences the phosphorylation of cell cycle related protein. However, the alteration of cell cycle protein could not lead to cell cycle change. 5. MAPK activation and cytoskeletal alteration are probably the early events induced by MC-LR in HL7702 cells.